Development of a real-time PCR assay for the detection and quantification of red Seabream iridovirus (RSIV)

A novel real-time PCR detection and quantification method for the red seabream iridovirus (RSIV) was developed using the major capsid protein (MCP) gene. A linear relationship was observed between the amount of input DNA and the threshold cycle (C-T) values over a range of 1 to 10(9) viral copies. The real-time PCR was 2 orders of magnitude more sensitive than the conventional PCR. It was also shown to be specific to RSIV, i.e., it did not react with either hirame rhabdovirus; (HIRRV), viral hemorrhagic septicemia virus (VHSV) or a DNA sample from apparently healthy red seabream Pagrus major. The viral load of dead juvenile red seabream (11 g average body weight) experimentally challenged with RSIV was in the range of 2.4 x 10(5) - 5.4 x 10(7) MCp gene copy number. To determine the target tissues of RSIV in the infected fish, 2 red seabream with average body weight of 50 g were experimentally challenged with RSIV and DNA was extracted from the various tissues and organs at 3rd day post-infection. Highest viral load was obtained in the heart with a mean MCP gene copy number of 2.7 x 10(5), while the blood had the lowest viral load at 8.8 x 10(3) mean MCP gene copy number. After the viral challenge, the real-time PCR assay was still able to detect RSIV from vaccinated survivors, which otherwise tested negative for RSIV by conventional PCR.